Plant cell proliferation is restricted to specialized regions called meristems. Stem cell populations within meristems give rise to all tissues of the adult plant. Cells generated in the shoot meristems are eventually incorporated into above ground lateral organs. In order to prevent depletion of shoot meristems, the loss of cells to lateral organs is balanced with the rate of stem cell proliferation. Mutations in the CLAVATA loci disrupt this balance and lead to a hyper-accumulation of stem cells, extra organ production and a reduction in fertility and plant growth. CLAVATA1 (CLV1) is a conserved receptor-like kinase (RLK) defined first in Arabidopsis and proposed to act as a receptor for the secreted peptide CLV3. After 20 years of genetic analysis we still do not know how CLV1 functions at the cellular level. Genetic evidence suggests that CLV1 signaling is regulated by CLV3-mediated internalization (LME). LME is an important mechanism for attenuating RLK signaling in plants and animals. Understanding CLV1 LME at the molecular level would allow us to address where in the meristem CLV1 is active, how CLV1 activation is regulated by other potential signaling partners and how CLV1 might activate downstream pathways that regulate stem cell production. Recent advances in live imaging in the Meyerowitz lab now allow this to be dissected. This proposal aims to understand CLV1 LME at the cellular level in Arabidopsis. In specific, an active CLV1 YFP fusion protein will be used to address where CLV1 is localized in the cell and how localization is regulated to CLV3 perception. This will be examined by using clv3 mutants, exogenous CLV3 peptides and known inhibitors of LME. The requirement for different domains of CLV1 in LME will be addressed using mutant versions of CLV1 -YFP . In addition, a system for creating conditionally inhibitable kinase versions of CLV1 will be detailed which will allow us to test if CLV3 activates CLV1 directly and will provide a tool to explore the effects of CLV1 inhibition in real time in live tissue. Lastly, I will detail a genetic strategy to identify mutants in key regulators of CLV1 LME which have so far proved elusive. This research will greatly expand our knowledge of RLK activation in plants and will serve as a model for studying signaling by other CLV1 or CLV3 family members. The work will provide our first knowledge of how CLV1 functions at the cellular level, a key roadblock for understanding how stem cell pathways are regulated in plants. This work will enlighten our knowledge of how this kingdom differs in stem cell biology from humans and other animals. This knowledge may lead to novel approaches for studying stem cell biology in general and may lead to novel approaches to treating disease in humans. [unreadable] [unreadable] [unreadable]